PROJECT SUMMARY Toxoplasma gondii is an intracellular parasite that chronically infects many hosts, including humans. Chronic infection requires T. gondii switching from a fast growing form to a slower encysted form. In humans, this life- long persistence primarily occurs in the brain where T. gondii can reactivate in the setting of acquired immune deficiencies. In AIDS patients, toxoplasmic encephalitis is the most common cause of focal brain lesions. Re- cent studies also suggest that persistent T. gondii infection may adversely affect cognition and global immune responses even in HIV+ patients on effective antiretroviral therapy. Currently, we have no drugs that target the encysted form of T. gondii. The goal of this grant is to develop new options for cyst-targeted anti-microbials by building upon our novel finding that ROP16, a parasite protein that is secreted into host cells early during inva- sion and that varies between T. gondii strains, affects encystment in a strain-specific manner. Among the ca- nonical encysting T. gondii strains? type II and III? the type III allele of ROP16 (ROP16III) phosphorylates STAT3 and 6, and possibly STAT5, while the type II allele (ROP16II) does not. In two different in vitro encyst- ment assays, we determined that a type III strain that lack ROP16 (III?rop16) has a 65% decrease in encyst- ment, while a type II strain that lacks ROP16 (II?rop16) has a mild increase in encystment. In host cells co- infected with a wild-type type III strain, but not a type II strain, the III?rop16 strain now shows normal encyst- ment. As there is no evidence that parasites re-uptake secreted effector proteins, this ?trans? complementation suggests that ROP16-dependent encystment depends upon host cell manipulations. We hypothesize, there- fore, that efficient type III encystment depends upon ROP16III manipulations of host cell signaling. To test this hypothesis, we will determine which properties of ROP16III are essential for ROP16-dependent encystment by complementing the III?rop16 strain with rop16III variants that lack nuclear localization, kinase activity, or STAT binding (Aim 1). To identify the host cell genes and pathways pertinent to ROP16-dependent encystment, we will use RNA-seq to perform transcriptional analysis of fibroblasts infected with the parental type III, III?rop16, or the complemented strain, and exposed to encystment conditions (Aim 2). Top differentially expressed tran- scripts/genes will be validated by Q-PCR and, when possible, immunofluorescent assays or western blots. With the completion of these aims, we will have determined which functions of ROP16III are essential for type III encystment and identified the host transcripts and pathways specifically affected by ROP16III in the context of encystment. These outcomes will establish a foundation on which to build long-term, mechanistic studies to define strain-specific mechanisms for T. gondii encystment. The work proposed here represents an important first step toward developing strain-specific treatments for the persistent form of T. gondii.